Figure 6: NMR structure of diSUMO-2 in complex with RNF4-SIM2,3.

(a) Plot of intensity difference between spectra for SUMO-2 dimers in complex with spin-labelled SIM2,3 peptide compared with the spectra in which MTSL is reduced by ascorbic acid. Data for complexes in which the distal subunit SUMO-2 ΔN11 is labelled are presented on the bottom row, whereas those for labelled proximal subunit SUMO-2 ΔGG are shown on the top. Data for complexes with spin-labelled SIM2,3 with MTSL positioned at C terminus are shown on the left and with MTSL at the N terminus on the right. Peak broadening (indicated by an intensity drop) is observed on the canonical SIM-binding site on β2 strand for both domains whether the peptide is N- or C-terminally spin-labelled. This suggests that the binding direction of the SIM2,3 peptide (N- to C terminus) relative to the SUMO chain (distal to proximal) is exchangeable. There are differences within the α-helix whether the C terminus of SIM2,3 is spin-labelled (that is, near SIM3) or at the N terminus (near SIM2). This indicates that the actual binding mode of SIM3 is distinct from SIM2, in which the helix is largely unaffected (right). (b) The NMR ensemble of the lowest energy 10 NMR structures for the complex of SUMO-2 dimer with SIM2,3 in which the proximal subunits (SUMO-2 ΔGG) are superposed. (c) The NMR ensemble of the lowest energy 10 structures for the complex of SUMO-2 dimer with SIM2,3 in which the distal subunits (SUMO-2 ΔN11) are superposed. The orientations showed on the right represent a 90° rotation from that on the left. The location of the N terminus of the SIM peptide is indicated by a sphere to illustrate its bi-directionality.